The long-term objectives of this research are to develop biodegradable scaffold systems that can direct stem cell differentiation, for the purposes of tissue regeneration. Stem cells hold great potential as cell sources for regenerative medicine. To be broadly useful therapeutically, methods must be developed to direct and control stem cell differentiation. It is clear that genetic signals can promote lineage-specific differentiation. The major barrier to gene delivery to stem cells is the lack of safe and efficient DMA delivery methods. I hypothesized that the sustained delivery of DMA condensed with biodegradable polymers from 3D scaffolds could be developed to direct stem cell differentiation. Accordingly, the following specific aims will be investigated: Aim 1. To develop safe and effective gene delivery systems to stem cells through synthesis and characterization of a large library of highly transfecting, poly((3-amino ester)s using high throughput approach. The library will be screened with the goals of 1) identifying highly effective gene delivery polymers to stem cells under serum containing conditions and 2) examining structure/function relationships to guide further polymer development. Promising vectors will be optimized for high efficacy and low cytotoxicity. Aim 2. To develop a fully biodegradable three-dimensional scaffold system that can deliver leading transfecting polymer/DNA nanoparticles identified from Aim 1. Methods will be developed to prepare dry- powder formulation of polymer/DNA nanoparticles and to encapsulate them into PGA/PLGA/PLA scaffolds using gas foaming approach. Aim 3. To evaluate in vivo gene delivery to stem cells and effects on bone regeneration via delivery of polymer/DNA nanoparticles from biodegradable polymer scaffolds developed in Aim 2. Specifically, stem cells will be seeded into scaffolds containing condensed polymer/DNA nanoparticles of bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF). The constructs will be implanted both subcutaneously in mice and in a cranial defect model in rats to evaluate bone formation. Outcome will be measured by radiography and histology. Organ failure and tissue damage are huge medical problems. Work from the proposed research would significantly improve surgical options by providing an alternative source for tissue augmentation using stem- cell based engineered tissues. The full biodegradability and biocompatibility of the proposed systems will make them readily translatable to clinics. [unreadable] [unreadable] [unreadable]